Graduate Thesis Or Dissertation

 

Oligoribonucleotides directed at ribosomal protein binding sites as inhibitors of ribosomal small subunit assembly and as possible antibiotics Public Deposited

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  • The effects of oligoribonucleotides of identical or similar sequences to a ribosomal protein binding site, specifically S8, on prokaryotic 30S ribosomal assembly were investigated in Escherichia coli. The oligoribonucleotides were expected to bind to the target protein S8 and block its incorporation into the 30S subunit, resulting in a nonfunctional particle. 30S assembly has been proposed to occur by similar pathways both in vivo and in vitro. If this block in assembly by the oligoribonucleotides occurs in vitro, it would also occur in vivo and could possibly lead to new modes of antibiotic therapy. The binding site of S8 was delivered into E. coli through a phagemid delivery system. The S8 binding site was cloned into a PBS+ vector under control of a lacZ promoter. The same phagemid with the ribosomal binding site of the lacZ gene deleted (the Shine-Dalgarno sequence and translational start site deleted) was also cloned and both transformed into E. coli. Growth experiments were performed in the presence and absence of the inducer isopropylthiogalactoside (IPTG). No change in growth upon induction of the phagemids was observed. Northern blot analysis was performed on cell lysates. By using a probe complementary to the S8 binding site and a probe 3' to the cloned S8 binding site, specific to the lacZ message, S8 binding site message from PBS-S8 was observed but not in amounts comparable to those of 16S rRNA. The phagemid with the deleted ribosomal binding site produced no message corresponding to either the S8 binding site or lacZ mRNA, possibly due to endonucleolytic attack upon the message. Omission of protein S8 in an in vitro reconstitution reaction results in subunits that show an 87-98% reduction in phenylalanine incorporation in the polyphenylalanine synthesis assay. This severe reduction of function was also used to test the hypothesis. Reconstitutions were performed in the presence of three unmodified oligoribonucleotides (CT4, CT10, CT11) identical or similar in sequence to the S8 binding site and assayed. None of the oligoribonucleotides showed a reduction of function when added at up to a 10:1 ratio of oligoribonucleotide to protein. CT11 was assayed to a 20:1 ratio of oligoribonucleotide to 30S proteins (TP30) and showed a 20% decrease of function at that ratio. Filter-binding assays were performed to test whether the oligoribonucleotides were binding to a protein in the TP30 mixture. The oligoribonucleotides bind protein but at much higher protein concentrations than 16S rRNA, signifying a lower affinity for the oligoribonuc1eotides. These data suggest that the oligoribonucleotides are binding to a protein but not with high enough affinity to compete with 16S rRNA. The concentration needed to be affective as an antibiotic would be too great to be practical.
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